The p21 proteins, the onc gene products of Harvey and Kirsten murine sarcoma viruses, are directly responsible for the virus-induced malignant transformation of the infected cells. As a step toward understanding the molecular mechanism of transformation, the biochemical properties of the p21 proteins were studied. The complete amino acid sequence of the p21 has been determined by two means: (1) DNA sequencing of the p21 coding region; (2) confirmation of the predicted sequence by direct amino acid sequencing of peptides derived from radio-labeled p21 protein. The p21 of Ha-MuSV possesses GTP-binding and autophosphorylation activities. By comparing the in vivo and in vitro sites of phosphorylation, it is concluded that the in vitro enzyme activity is responsible for the in vivo p21 phosphorylation. The amino acid sequences surrounding the phosphorylation site has been determined by sequencing the phosphopeptides. Both the Harvey and the Kirsten p21s share the same phosphorylation site.